Methods of administering anti-TNFalpha antibodies

ABSTRACT

Methods of treating disorders in which TFNα activity is detrimental via biweekly, subcutaneous administration of human antibodies, preferably recombinant human antibodies, that specifically bind to human tumor necrosis factor α (hTNFα) are disclosed. The antibody may be administered with or without methotrexate. These antibodies have high affinity for hTNFα (e.g., K d =10 −8  M or less), a slow off rate for hTNFα dissociation (e.g., K off =10 −3  sec −1  or less) and neutralize hTNFα activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. Kits containing a pharmaceutical composition and instructions for dosing, and preloaded syringes containing pharmaceutical compositions are also encompassed by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/296,961, filed Jun. 8, 2001.

BACKGROUND OF THE INVENTION

[0002] Tumor necrosis factor α (TNFα) is a cytokine produced by numerouscell types, including monocytes and macrophages, that was originallyidentified based on its capacity to induce the necrosis of certain mousetumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, afactor termed cachectin, associated with cachexia, was shown to be thesame molecule as TNFα. TNFα has been implicated in mediating shock (seee.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655).Furthermore, TNFα has been implicated in the pathophysiology of avariety of other human diseases and disorders, including sepsis,infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Vasilli, P. (1992) Annu. Rev.Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med.45:491-503).

[0003] Because of the harmful role of human TNFα ( (hTNFα) in a varietyof human disorders, therapeutic strategies have been designed to inhibitor counteract hTNFα activity. In particular, antibodies that bind to,and neutralize, hTNFα have been sought as a means to inhibit hTNFαactivity. Some of the earliest of such antibodies were mouse monoclonalantibodies (mAbs), secreted by hybridomas prepared from lymphocytes ofmice immunized with hTNFα (see e.g., Hahn T; et al., (1985) Proc NatlAcad Sci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys.Res. Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Möller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 186 833 B1 by Wallach, D.; EuropeanPatent Application Publication No. 218 868 A1 by Old et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A., et al.). While thesemouse anti-hTNFα antibodies often displayed high affinity for hTNFα(e.g., Kd≦10⁻⁹M) and were able to neutralize hTNFα activity, their usein vivo may be limited by problems associated with administration ofmouse antibodies to humans, such as short serum half life, an inabilityto trigger certain human effector functions and elicitation of anunwanted immune response against the mouse antibody in a human (the“human antimouse antibody” (HAMA) reaction).

[0004] In an attempt to overcome the problems associated with use offully-murine antibodies in humans, murine anti-hTNFα antibodies havebeen genetically engineered to be more “human-like.” For example,chimeric antibodies, in which the variable regions of the antibodychains are murine-derived and the constant regions of the antibodychains are human-derived, have been prepared (Knight, D. M, et al.(1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 byDaddona, P. E., et al.). Additionally, humanized antibodies, in whichthe hypervariable domains of the antibody variable regions aremurine-derived but the remainder of the variable regions and theantibody constant regions are human-derived, have also been prepared(PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods, e.g., for chronic indications, such as rheumatoidarthritis (see e.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127;Elliot, M. J., et al. (1994) Lancet 344:1105-1110).

[0005] A preferred hTNFα inhibitory agent to murine mAbs or derivativesthereof (e.g., chimeric or humanized antibodies) would be an entirelyhuman anti-hTNFα antibody, since such an agent should not elicit theHAMA reaction, even if used for prolonged periods. Human monoclonalautoantibodies against hTNFα have been prepared using human hybridomatechniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; Boyle,P., et al. (1993) Cell. Immunol. 152:569-581; European PatentApplication Publication No. 614 984 A2 by Boyle, et al.). However, thesehybridoma-derived monoclonal autoantibodies were reported to have anaffinity for hTNFα that was too low to calculate by conventionalmethods, were unable to bind soluble hTNFα and were unable to neutralizehTNFα-induced cytotoxicity (see Boyle, et al.; supra). Moreover, thesuccess of the human hybridoma technique depends upon the naturalpresence in human peripheral blood of lymphocytes producingautoantibodies specific for hTNFα. Certain studies have detected serumautoantibodies against hTNFα in human subjects (Fomsgaard, A., et al.(1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog.Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., etal. (1991) J. Immunol. Methods 139:145-147).

[0006] Alternative to naturally-occurring human anti-hTNFα antibodieswould be a recombinant hTNFα antibody. Recombinant human antibodies thatbind hTNFα with relatively low affinity (i.e., K_(d)˜10⁻⁷M) and a fastoff rate (i.e., K_(off)˜10⁻² sec⁻¹) have been described (Griffiths, A.D., et al. (1993) EMBO J. 12:725-734). However, because of theirrelatively fast dissociation kinetics, these antibodies may not besuitable for therapeutic use. Additionally, a recombinant humananti-hTNFα has been described that does not neutralize hTNFα activity,but rather enhances binding of hTNFα to the surface of cells andenhances internalization of hTNFα (Lidbury, A., et al. (1994)Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R.et al.)

[0007] Recombinant human antibodies that bind soluble hTNFα with highaffinity and slow dissociation kinetics and that have the capacity toneutralize hTNFα activity, including hTNFα-induced cytotoxicity (invitro and in vivo) and hTNFα-induced cell activation, have also beendescribed (see U.S. Pat. No. 6,090,382). Typical protocols foradministering antibodies are performed intravenously on a weekly basis.Weekly dosing with antibodies and/or any drug can be costly, cumbersome,and result in an increase in the number of side effects due to thefrequency of administration. Intravenous administration also haslimitations in that the administration is usually provided by someonewith medical training.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods for biweekly dosingregimens for the treatment of TNFα associated disorders, preferably viaa subcutaneous route. Biweekly dosing has many advantages over weeklydosing including, but not limited to, a lower number of totalinjections, decreased number of injection site reactions (e.g., localpain and swelling), increased patient compliance (i.e., due to lessfrequent injections), and less cost to the patient as well as the healthcare provider. Subcutaneous dosing is advantageous because the patientmay self-administer a therapeutic substance, e.g., a human TNFαantibody, which is convenient for both the patient and the health careprovider.

[0009] This invention provides methods for treating disorders in whichTNFα activity is detrimental. The methods include administeringbiweekly, subcutaneous injections of antibodies to a subject. Theantibodies preferably are recombinant human antibodies that specificallybind to human TNFα. This invention further provides methods for treatingdisorders in which TNFα activity is detrimental. These methods includeutilizing a combination therapy wherein human antibodies areadministered to a subject with another therapeutic agent, such as one ormore additional antibodies that bind other targets (e.g., antibodiesthat bind other cytokines or that bind cell surface molecules), one ormore cytokines, soluble TNFα receptor (see e.g., PCT Publication No. WO94/06476) and/or one or more chemical agents that inhibit hTNFαproduction or activity (such as cyclohexane-ylidene derivatives asdescribed in PCT Publication No. WO 93/19751), preferably methotrexate.The antibodies are preferably recombinant human antibodies thatspecifically bind to human TNFα. The antibodies of the invention arecharacterized by binding to hTNFα with high affinity and slowdissociation kinetics and by neutralizing hTNFα activity, includinghTNFα-induced cytotoxicity (in vitro and in vivo) and hTNFα-inducedcellular activation. The antibodies can be full-length (e.g., an IgG1 orIgG4 antibody) or can comprise only an antigen-binding portion (e.g., aFab, F(ab′)₂, scFv fragment or single domain). The most preferredrecombinant antibody of the invention, termed D2E7, has a light chainCDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and aheavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:4 (set forth in Appendix B). Preferably, the D2E7 antibody has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2. These antibodies are described in U.S.Pat. No. 6,090,382, incorporated in its entirety herein by reference.

[0010] In one embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental. These methods includeinhibiting human TNFα activity by subcutaneous, biweekly administrationof an anti-TNFα antibody such that the disorder is treated. The disordercan be, for example, sepsis, an autoimmune disease (e.g., rheumatoidarthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmuneuveitis and nephrotic syndrome), an infectious disease, a malignancy,transplant rejection or graft-versus-host disease, a pulmonary disorder,a bone disorder, an intestinal disorder or a cardiac disorder.

[0011] In another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental. These methods includeinhibiting human TNFα activity by subcutaneous administration of ananti-TNFα antibody and methotrexate such that the disorder is treated.In one aspect, methotrexate is administered together with an anti-TNFαantibody. In another aspect, methotrexate is administered prior to theadministration of an anti-TNFα antibody. In still another aspect,methotrexate is administered subsequent to the administration of ananti-TNFα antibody.

[0012] In a preferred embodiment, the anti-TNFα antibody used to treatdisorders in which TNFα activity is detrimental is a human anti-TNFαantibody. Even more preferably, treatment occurs by the biweekly,subcutaneous administration of an isolated human antibody, or anantigen-binding portion thereof. The antibody or antigen-binding portionthereof preferably dissociates from human TNFα with a K_(d) of 1×10⁻⁸ Mor less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less. More preferably, the isolated human antibody, orantigen-binding portion thereof, dissociates from human TNFα with aK_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, with a K_(off)of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated human antibody, orantigen-binding portion thereof, neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less, evenmore preferably with an IC₅₀ of 1×10⁻⁹ M or less and still morepreferably with an IC₅₀ of 1×10³¹ ¹⁰ M or less.

[0013] In another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental by the biweekly,subcutaneous administration to the subject a human antibody, orantigen-binding portion thereof. The antibody or antigen-binding portionthereof preferably has the following characteristics:

[0014] a) dissociates from human TNFα with a K_(off) of 1×10⁻³ s⁻¹ orless, as determined by surface plasmon resonance;

[0015] b) has a light chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8 or by one to fiveconservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9;

[0016] c) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12.

[0017] More preferably, the antibody, or antigen-binding portionthereof, dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ orless. Still more preferably, the antibody, or antigen-binding portionthereof, dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ orless.

[0018] In yet another embodiment, the invention provides methods oftreating disorders in which TNFα activity is detrimental. These methodsinclude a biweekly, subcutaneous administration to the subject a humanantibody, or an antigen-binding portion thereof. The antibody orantigen-binding portion thereof preferably contains an LCVR having CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, and with an HCVR having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Morepreferably, the LCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 6. Still morepreferably, the LCVR further has CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7 and the HCVR has a CDR1 domain comprising theamino acid sequence of SEQ ID NO: 8.

[0019] In still another embodiment, the invention provides methods oftreating disorders in which TNFα activity is detrimental bysubcutaneously administering to the subject, biweekly, an isolated humanantibody, or an antigen binding portion thereof. The antibody orantigen-binding portion thereof preferably contains an LCVR comprisingthe amino acid sequence of SEQ ID NO: 1 and an HCVR comprising the aminoacid sequence of SEQ ID NO: 2. In certain embodiments, the antibody hasan IgG1 heavy chain constant region or an IgG4 heavy chain constantregion. In yet other embodiments, the antibody is a Fab fragment, anF(ab′)₂ fragment or a single chain Fv fragment.

[0020] In still other embodiments, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial by subcutaneously administering to the subject, biweekly,one or more anti-TNFα antibodies, or antigen-binding portions thereof.The antibody or antigen-binding portion thereof preferably contains anLCVR having CDR3 domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or withan HCVR having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34 and SEQ ID NO: 35.

[0021] Still another aspect of the invention pertains to kits containinga formulation comprising a pharmaceutical composition. The kits comprisean anti-TNFα antibody and a pharmaceutically acceptable carrier. Thekits contain instructions for biweekly subcutaneous dosing of thepharmaceutical composition for the treatment of a disorder in which theadministration of an anti-TNFα antibody is beneficial. In anotheraspect, the invention pertains to kits containing a formulationcomprising a pharmaceutical composition, further comprising an anti-TNFαantibody, methotrexate, and a pharmaceutically acceptable carrier. Thekits contain instructions for subcutaneous dosing of the pharmaceuticalcomposition for the treatment of a disorder in which the administrationof an anti-TNFα antibody is beneficial.

[0022] Still another aspect of the invention provides a preloadedsyringe containing a pharmaceutical composition comprising an anti-TNFαantibody and a pharmaceutically acceptable carrier. In still anotheraspect, the invention provides a preloaded syringe containing apharmaceutical composition comprising an anti-TNFα antibody,methotrexate, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1A and 1B depict the American College of Rheumatology 20(ACR20) and ACR50 responses for patients suffering from rheumatoidarthritis (RA) after subcutaneous dosing with the antibody D2E7 everyweek for a total of twelve weeks (1A), or subcutaneous dosing with theantibody D2E7 and methotrexate every other week (1B) for a total oftwenty-four weeks. These data indicate that every other week dosing isas effective as every week dosing.

[0024]FIG. 2 depicts ACR20, ACR50, and ACR70 responses for patientssuffering from RA after subcutaneous dosing with the antibody D2E7 andmethotrexate every other week at twenty-four weeks.

[0025]FIGS. 3A and 3B depict time courses of tender joint count (3A) andswollen joint count (3B) over twenty-four weeks for patients sufferingfrom RA after subcutaneous dosing with D2E7 and methotrexate every otherweek at twenty-four weeks.

[0026]FIG. 4 depicts results from a short form health survey (SF-36)from patients suffering from RA after subcutaneous dosing with theantibody D2E7 and methotrexate every other week at twenty-four weeks.RP, role physical; PF, physical function; BP, bodily pain; GH, generalhealth; V, vitality; SF, social functioning; RE, role emotional; and ME,mental health.

[0027]FIG. 5 depicts the percentage of ACR responders following a singleintravenous injection of the antibody D2E7 and methotrexate in patientssuffering from RA.

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention pertains to methods of treating disorders in whichthe administration of an anti-TNFα antibody is beneficial comprising theadministration of isolated human antibodies, or antigen-binding portionsthereof, that bind to human TNFα with high affinity, a low off rate andhigh neutralizing capacity such that the disorder is treated. Variousaspects of the invention relate to treatment with antibodies andantibody fragments, and pharmaceutical compositions thereof.

[0029] In order that the present invention may be more readilyunderstood, certain terms are first defined.

[0030] The term “dosing”, as used herein, refers to the administrationof a substance (e.g., an anti-TNFα antibody) to achieve a therapeuticobjective (e.g., the treatment of a TNFα-associated disorder).

[0031] The terms “biweekly dosing regimen”, “biweekly dosing”, and“biweekly administration”, as used herein, refer to the time course ofadministering a substance (e.g., an anti-TNFα antibody) to a subject toachieve a therapeutic objective (e.g., the treatment of aTNFα-associated disorder). The biweekly dosing regimen is not intendedto include a weekly dosing regimen. Preferably, the substance isadministered every 9-19 days, more preferably, every 11-17 days, evenmore preferably, every 13-15 days, and most preferably, every 14 days.

[0032] The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-αantibody and the drug methotrexate. The methotrexate may be administeredconcomitant with, prior to, or following the administration of ananti-TNFα antibody.

[0033] The term “human TNFα” (abbreviated herein as hTNFα, or simplyhTNF), as used herein, is intended to refer to a human cytokine thatexists as a 17 kD secreted form and a 26 kD membrane associated form,the biologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of TNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E.Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

[0034] The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0035] The term “antigen-binding portion” of an antibody (or simply“antibody portion”), as used herein, refers to one or more fragments ofan antibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

[0036] Still further, an antibody or antigen-binding portion thereof maybe part of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

[0037] The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

[0038] The term “recombinant human antibody”, as used herein, isintended to include all human antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies expressedusing a recombinant expression vector transfected into a host cell(described further in Section II, below), antibodies isolated from arecombinant, combinatorial human antibody library (described further inSection III, below), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

[0039] An “isolated antibody”, as used herein, is intended to refer toan antibody that is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds hTNFα is substantially free of antibodies thatspecifically bind antigens other than hTNFα). An isolated antibody thatspecifically binds hTNFα may, however, have cross-reactivity to otherantigens, such as hTNFα molecules from other species (discussed infurther detail below). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

[0040] A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see Example 4). Preferably, the ability of an antibody toneutralize hTNFα activity is assessed by inhibition of hTNFα-inducedcytotoxicity of L929 cells. As an additional or alternative parameter ofhTNFα activity, the ability of an antibody to inhibit hTNFα-inducedexpression of ELAM-1 on HUVEC, as a measure of hTNFα-induced cellularactivation, can be assessed.

[0041] The term “surface plasmon resonance”, as used herein, refers toan optical phenomenon that allows for the analysis of real-timebiospecific interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIAcoresystem (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).For further descriptions, see Example 1 and Jönsson, U., et al. (1993)Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0042] The term “K_(off)”, as used herein, is intended to refer to theoff rate constant for dissociation of an antibody from theantibody/antigen complex.

[0043] The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

[0044] The term “nucleic acid molecule”, as used herein, is intended toinclude DNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

[0045] The term “isolated nucleic acid molecule”, as used herein inreference to nucleic acids encoding antibodies or antibody portions(e.g., VH, VL, CDR3) that bind hTNFα, is intended to refer to a nucleicacid molecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than hTNFα,which other sequences may naturally flank the nucleic acid in humangenomic DNA. Thus, for example, an isolated nucleic acid of theinvention encoding a VH region of an anti-hTNFα antibody contains noother sequences encoding other VH regions that bind antigens other thanhTNFα.

[0046] The term “vector”, as used herein, is intended to refer to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Another type of vector is a viral vector,wherein additional DNA segments may be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”). Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. In the present specification, “plasmid”and “vector” may be used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

[0047] The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

[0048] Various aspects of the invention are described in further detailin the following subsections.

[0049] I. Human Antibodies that Bind Human TNFα

[0050] This invention provides methods of treating disorders in whichthe administration of an anti-TNFα antibody is beneficial. These methodsinclude the biweekly, subcutaneous administration of isolated humanantibodies, or antigen-binding portions thereof, that bind to human TNFαwith high affinity, a low off rate and high neutralizing capacity.Preferably, the human antibodies of the invention are recombinant,neutralizing human anti-hTNFα antibodies. The most preferredrecombinant, neutralizing antibody of the invention is referred toherein as D2E7 (the amino acid sequence of the D2E7 VL region is shownin SEQ ID NO: 1; the amino acid sequence of the D2E7 VH region is shownin SEQ ID NO: 2). The properties of D2E7 have been described in Salfeldet al., U.S. Pat. No. 6,090,382, which is incorporated by referenceherein.

[0051] In one aspect, the invention pertains to treating disorders inwhich the administration of an anti-TNFα antibody is beneficial. Thesetreatments include the biweekly, subcutaneous administration of D2E7antibodies and antibody portions, D2E7-related antibodies and antibodyportions, and other human antibodies and antibody portions withequivalent properties to D2E7, such as high affinity binding to hTNFαwith low dissociation kinetics and high neutralizing capacity. In oneembodiment, the invention provides treatment with an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhuman TNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 1×10⁻¹⁰ M or less. In a preferredembodiment, the antibody is an isolated human recombinant antibody, oran antigen-binding portion thereof.

[0052] It is well known in the art that antibody heavy and light chainCDR3 domains play an important role in the binding specificity/affinityof an antibody for an antigen. Accordingly, in another aspect, theinvention pertains to methods of treating disorders in which theadministration of an anti-TNFα antibody is beneficial by subcutaneousadministration of human antibodies that have slow dissociation kineticsfor association with hTNFα and that have light and heavy chain CDR3domains that structurally are identical to or related to those of D2E7.Position 9 of the D2E7 VL CDR3 can be occupied by Ala or Thr withoutsubstantially affecting the K_(off). Accordingly, a consensus motif forthe D2E7 VL CDR3 comprises the amino acid sequence:Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3 ). Additionally, position 12 of theD2E7 VH CDR3 can be occupied by Tyr or Asn, without substantiallyaffecting the K_(off). Accordingly, a consensus motif for the D2E7 VHCDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQID NO: 4). Moreover, as demonstrated in Example 2, the CDR3 domain ofthe D2E7 heavy and light chains is amenable to substitution with asingle alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3 )without substantially affecting the K_(off). Still further, the skilledartisan will appreciate that, given the amenability of the D2E7 VL andVH CDR3 domains to substitutions by alanine, substitution of other aminoacids within the CDR3 domains may be possible while still retaining thelow off rate constant of the antibody, in particular substitutions withconservative amino acids. A “conservative amino acid substitution”, asused herein, is one in which one amino acid residue is replaced withanother amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Preferably, no more than one to five conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains. Morepreferably, no more than one to three conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains.Additionally, conservative amino acid substitutions should not be madeat amino acid positions critical for binding to hTNFα. Positions 2 and 5of the D2E7 VL CDR3 and positions 1and 7 of the D2E7 VH CDR3 appear tobe critical for interaction with hTNFα and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above) (see U.S. Pat. No. 6,090,382).

[0053] Accordingly, in another embodiment, the invention providesmethods of treating disorders in which the administration of ananti-TNFα antibody is beneficial by the biweekly, subcutaneousadministration of an isolated human antibody, or antigen-binding portionthereof. The antibody or antigen-binding portion thereof preferablycontains the following characteristics:

[0054] a) dissociates from human TNFα with a K_(off) rate constant of1×10⁻s⁻¹ or less, as deternined by surface plasmon resonance;

[0055] b) has a light chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8 or by one to fiveconservative amino acid substitutions at positions 1 , 3, 4, 6, 7, 8and/or 9;

[0056] c) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12.

[0057] More preferably, the antibody, or antigen-binding portionthereof, dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ orless. Even more preferably, the antibody, or antigen-binding portionthereof, dissociates from human, TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ orless.

[0058] In yet another embodiment, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial by the biweekly, subcutaneous administration of anisolated human antibody, or an antigen-binding portion thereof. Theantibody or antigen-binding portion thereof preferably contains a lightchain variable region (LCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chainvariable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.Preferably, the LCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and the HCVR furtherhas a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6(i.e., the D2E7 VH CDR2). Even more preferably, the LCVR further hasCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e.,the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The frameworkregions for VL preferably are from the V_(κ)I human germline family,more preferably from the A20human germline Vk gene and most preferablyfrom the D2E7 VL framework sequences shown in FIGS. 1A and 1B of U.S.Pat. No. 6,090,382. The framework regions for VH preferably are from theVH3 human germline family, more preferably from the DP-31 human germlineVH gene and most preferably from the D2E7 VH framework sequences shownin FIGS. 2A and 2B U.S. Pat. No. 6,090,382.

[0059] In still another embodiment, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial by the biweekly, subcutaneous administration of anisolated human antibody, or an antigen binding portion thereof. Theantibody or antigen-binding portion thereof preferably contains a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 (i.e., the D2E7 VL) and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the D2E7 VH).In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgGl heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

[0060] In still other embodiments, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial by the biweekly, subcutaneous administration of anisolated human antibody, or an antigen-binding portions thereof. Theantibody or antigen-binding portion thereof preferably containsD2E7-related VL and VH CDR3 domains, for example, antibodies, orantigen-binding portions thereof, with a light chain variable region(LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 orwith a heavy chain variable region (HCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQID NO: 35.

[0061] An antibody or antibody portion of the invention can bederivatized or linked to another functional molecule (e.g., anotherpeptide or protein). Accordingly, the antibodies and antibody portionsof the invention are intended to include derivatized and otherwisemodified forms of the human anti-hTNFα antibodies described herein,including immunoadhesion molecules. For example, an antibody or antibodyportion of the invention can be functionally linked (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

[0062] One type of derivatized antibody is produced by crosslinking twoor more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

[0063] Useful detectable agents with which an antibody or antibodyportion of the invention may be derivatized include fluorescentcompounds. Exemplary fluorescent detectable agents include fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, glucose oxidase and thelike. When an antibody is derivatized with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce adetectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

[0064] II. Expression of Antibodies

[0065] An antibody, or antibody portion, of the invention can beprepared by recombinant expression of immunoglobulin light and heavychain genes in a host cell. To express an antibody recombinantly, a hostcell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and, preferably, secreted into the medium inwhich the host cells are cultured, from which medium the antibodies canbe recovered. Standard recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397 by Boss et al.

[0066] To express D2E7 or a D2E7-related antibody, DNA fragmentsencoding the light and heavy chain variable regions are first obtained.These DNAs can be obtained by amplification and modification of germlinelight and heavy chain variable sequences using the polymerase chainreaction (PCR). Germline DNA sequences for human heavy and light chainvariable region genes are known in the art (see e.g., the “Vbase” humangermline sequence database; see also Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al. (1992) “The Repertoire of Human Germline V_(H)Sequences Reveals about Fifty Groups of V_(H) Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line V₇₈ Segments Reveals a StrongBias in their Usage” Eur. J. Immunol. 24:827-836; the contents of eachof which are expressly incorporated herein by reference). To obtain aDNA fragment encoding the heavy chain variable region of D2E7, or aD2E7-related antibody, a member of the V_(H)3 family of human germlineVH genes is amplified by standard PCR. Most preferably, the DP-31 VHgermline sequence is amplified. To obtain a DNA fragment encoding thelight chain variable region of D2E7, or a D2E7-related antibody, amember of the V_(κ)I family of human germline VL genes is amplified bystandard PCR. Most preferably, the A20 VL germline sequence isamplified. PCR primers suitable for use in amplifying the DP-31 germlineVH and A20 germline VL sequences can be designed based on the nucleotidesequences disclosed in the references cited supra, using standardmethods.

[0067] Once the germline VH and VL fragments are obtained, thesesequences can be mutated to encode the D2E7 or D2E7-related amino acidsequences disclosed herein. The amino acid sequences encoded by thegermline VH and VL DNA sequences are first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residuesin the D2E7 or D2E7-related sequence that differ from germline. Then,the appropriate nucleotides of the germline DNA sequences are mutatedsuch that the mutated gernline sequence encodes the D2E7 or D2E7-relatedamino acid sequence, using the genetic code to determine whichnucleotide changes should be made. Mutagenesis of the germline sequencesis carried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis.

[0068] Once DNA fragments encoding D2E7 or D2E7-related VH and VLsegments are obtained (by amplification and mutagenesis of germline VHand VL genes, as described above), these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker. The term “operatively linked”, as used in thiscontext, is intended to mean that the two DNA fragments are joined suchthat the amino acid sequences encoded by the two DNA fragments remainin-frame.

[0069] The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

[0070] The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

[0071] To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0072] To express the antibodies, or antibody portions of the invention,DNAs encoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the D2E7 or D2E7-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the D2E7 orD2E7-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

[0073] In addition to the antibody chain genes, the recombinantexpression vectors of the invention carry regulatory sequences thatcontrol the expression of the antibody chain genes in a host cell. Theterm “regulatory sequence” is intended to includes promoters, enhancersand other expression control elements (e.g., polyadenylation signals)that control the transcription or translation of the antibody chaingenes. Such regulatory sequences are described, for example, in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

[0074] In addition to the antibody chain genes and regulatory sequences,the recombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

[0075] For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

[0076] Preferred mammalian host cells for expressing the recombinantantibodies of the invention include Chinese Hamster Ovary (CHO cells)(including dhfr⁻ CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

[0077] Host cells can also be used to produce portions of intactantibodies, such as Fab fragments or scFv molecules. It will beunderstood that variations on the above procedure are within the scopeof the present invention. For example, it may be desirable to transfecta host cell with DNA encoding either the light chain or the heavy chain(but not both) of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to hTNFα. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than hTNFα by crosslinkingan antibody of the invention to a second antibody by standard chemicalcrosslinking methods.

[0078] In a preferred system for recombinant expression of an antibody,or antigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

[0079] III. Selection of Recombinant Human Antibodies

[0080] Recombinant human antibodies of the invention in addition to D2E7or an antigen binding portion thereof, or D2E7-related antibodiesdisclosed herein can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612), examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display libraries can be found in, for example,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Biolfechnology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

[0081] In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990 )348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFvantibody libraries preferably are screened using recombinant human TNFαas the antigen.

[0082] Once initial human VL and VH segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedVL and VH segments are screened for hTNFα binding, are performed toselect preferred VL/VH pair combinations. Additionally, to furtherimprove the affinity and/or lower the off rate constant for hTNFαbinding, the VL and VH segments of the preferred VL/VH pair(s) can berandomly mutated, preferably within the CDR3 region of VH and/or VL, ina process analogous to the in vivo somatic mutation process responsiblefor affinity maturation of antibodies during a natural immune response.This in vitro affinity maturation can be accomplished by amplifying VHand VL regions using PCR primers complimentary to the VH CDR3 or VLCDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode VH and VL segments into which randommutations have been introduced into the VH and/or VL CDR3 regions. Theserandomly mutated VH and VL segments can be rescreened for binding tohTNFα and sequences that exhibit high affinity and a low off rate forhTNFα binding can be selected.

[0083] Following screening and isolation of an anti-hTNFα antibody ofthe invention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in Section II above.

[0084] IV. Pharmaceutical Compositions and Pharmaceutical Administration

[0085] The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject for the methods described herein, e.g.,biweekly, subcutaneous dosing. Typically, the pharmaceutical compositioncomprises an antibody (or antibody portion) of the invention and/ormethotrexate and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible and are suitable for administration to asubject for the methods described herein. Examples of pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

[0086] The compositions of this invention may be in a variety of forms.These include, for example, liquid, semi-solid and solid dosage forms,such as liquid solutions (e.g., injectable and infusible solutions),dispersions or suspensions, tablets, pills, powders, liposomes andsuppositories. The preferred form depends on the intended mode ofadministration and therapeutic application. Typical preferredcompositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies. The preferred mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In a preferred embodiment, the antibody is administeredby intravenous infusion or injection. In another preferred embodiment,the antibody is administered by intramuscular injection. In aparticularly preferred embodiment, the antibody is administered bysubcutaneous injection (e.g., a biweekly, subcutaneous injection).

[0087] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

[0088] The antibodies and antibody-portions of the present invention canbe administered by a variety of methods known in the art, although formany therapeutic applications, the preferred route/mode ofadministration is subcutaneous injection. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyethylene glycol (PEG), polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

[0089] In certain embodiments, an antibody or antibody portion of theinvention may be orally administered, for example, with an inert diluentor an assimilable edible carrier. The compound (and other ingredients,if desired) may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the compounds maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

[0090] Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents. For example, an anti-hTNFα antibodyor antibody portion of the invention may be coformulated and/orcoadministered with methotrexate, one or more additional antibodies thatbind other targets (e.g., antibodies that bind other cytokines or thatbind cell surface molecules), one or more cytokines, soluble TNFαreceptor (see e.g., PCT Publication No. WO 94/06476) and/or one or morechemical agents that inhibit hTNFα production or activity (such ascyclohexaneylidene derivatives as described in PCT Publication No. WO93/19751). Furthermore, one or more antibodies of the invention may beused in combination with two or more of the foregoing therapeuticagents. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.The use of the antibodies, or antibody portions, of the invention incombination with other therapeutic agents is discussed further insubsection IV.

[0091] Non-limiting examples of therapeutic agents for rheumatoidarthritis with which an antibody, or antibody portion, of the inventioncan be combined include the following: non-steroidal anti-inflammatorydrug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s)(CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Inmunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatizedanti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis & Rheumatism(1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusionproteins; Seragen; see e.g., Arthritis & Rheumatism (1993) Vol. 36,1223); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF bindingprotein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S284; Amer. J. Physiol.-Heart and Circulatory Physiology(1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase Type IVinhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No.9 (supplement), S81); Iloprost (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S82);methotrexate; thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282) and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid(inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S284); T-614 (cytokine inhibitor;see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S282); prostaglandin E1 (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatorydrug; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; seee.g., Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam(non-steroidal antiinflammatory drug); Ibuprofen (non-steroidalanti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatorydrug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin(non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281);Azathioprine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); ICE inhibitor (inhibitor of the enzymeinterleukin-1β converting enzyme); zap-70 and/or lck inhibitor(inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/orVEGF-R inhibitor (inhibitos of vascular endothelial cell growth factoror vascular endothelial cell growth factor receptor; inhibitors ofangiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580);TNF-convertase inhibitors; anti-IL12 antibodies; interleukin-11 (seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S296);interleukin-13 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold;penicillamine; chloroquine; hydroxychloroquine; chlorambucil;cyclophosphamide; cyclosporine; total lymphoid irradiation;anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus(rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); and azaribine.

[0092] Non-limiting examples of therapeutic agents for inflammatorybowel disease with which an antibody, or antibody portion, of theinvention can be combined include the following: budenoside; epidermalgrowth factor; corticosteroids; cyclosporin, sulfasalazine;aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;anti-IL-1β monoclonal antibodies; anti-IL-6 monoclonal antibodies;growth factors; elastase inhibitors; pyridinyl-imidazole compounds;CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2(chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche);interleukin-10 (SCH 52000; Schering Plough); IL-4; IL-10 and/orIL-4-agonists (e.g., agonist antibodies); interleukin-11; glucuronide-or dextran-conjugated prodrugs of prednisolone, dexamethasone orbudesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides(ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1(TP10; T Cell Sciences, Inc.); slowrelease mesalazine; methotrexate;antagonists of Platelet Activating Factor (PAF); ciprofloxacin; andlignocaine.

[0093] Nonlimiting examples of therapeutic agents for multiple sclerosiswith which an antibody, or antibody portion, of the invention can becombined include the following: corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (Avonex™;Biogen); interferon-β1b (Betaseron™; Chiron/Berlex); Copolymer 1 (Cop-1;Copaxone™; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;intravenous immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody;Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein;Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche); IL-10; IL-4; and IL-10and/or IL-4 agonists (e.g., agonist antibodies).

[0094] Nonlimiting examples of therapeutic agents for sepsis with whichan antibody, or antibody portion, of the invention can be combinedinclude the following: hypertonic saline solutions; antibiotics;intravenous gamma globulin; continuous hemofiltration; carbapenems(e.g., meropenem); antagonists of cytokines such as TNFα, IL-1β, IL-6and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466(Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide;SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (PicowerInstitute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP(chemically modified hemoglobin; APEX Bioscience); iron chelators andchelates, including diethylenetriamine pentaacetic acid-iron (III)complex (DTPA iron (III); Molichem Medicines); lisofylline (syntheticsmall molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan(aqeuous soluble β1,3glucan; Alpha-Beta Technology); apolipoprotein A-1reconstituted with lipids; chiral hydroxamic acids (syntheticantibacterials that inhibit lipid A biosynthesis); anti-endotoxinantibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.);rBPI₂₁ (recombinant N-terminal fragment of humanBactericidal/Permeability-Increasing Protein); and SyntheticAnti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);

[0095] Nonlimiting examples of therapeutic agents for adult respiratorydistress syndrome (ARDS) with which an antibody, or antibody portion, ofthe invention can be combined include the following: anti-IL-8antibodies; surfactant replacement therapy; CDP-571/BAY-10-3356(humanized anti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFαantibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusionprotein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295;J. Invest. Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein; Hoffmann-LaRoche).

[0096] The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0097] Dosage regimens may be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response). For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0098] An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 10-100 mg, more preferably 20-80 mg and most preferablyabout 40 mg. It is to be noted that dosage values may vary with the typeand severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

[0099] V. Uses of the Antibodies of the Invention

[0100] Given their ability to bind to hTNFα, the anti-hTNFα antibodies,or portions thereof, of the invention can be used to detect hTNFα (e.g.,in a biological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The inventionprovides a method for detecting hTNFα in a biological sample comprisingcontacting a biological sample with an antibody, or antibody portion, ofthe invention and detecting either the antibody (or antibody portion)bound to hTNFα or unbound antibody (or antibody portion), to therebydetect hTNFα in the biological sample. The antibody is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

[0101] Alternative to labeling the antibody, hTNFα can be assayed inbiological fluids by a competition immunoassay utilizing rhTNFαstandards labeled with a detectable substance and an unlabeledanti-hTNFα antibody. In this assay, the biological sample, the labeledrhTNFα standards and the anti-hTNFα antibody are combined and the amountof labeled rhTNFα standard bound to the unlabeled antibody isdetermined. The amount of hTNFα in the biological sample is inverselyproportional to the amount of labeled hTNFα standard bound to theanti-hTNFα antibody.

[0102] A D2E7 antibody of the invention can also be used to detect TNFαsfrom species other than humans, in particular TNFαs from primates (e.g.,chimpanzee, baboon, marmoset, cynomolgus and rhesus), pig and mouse,since D2E7 can bind to each of these TNFαs.

[0103] The antibodies and antibody portions of the invention are capableof neutralizing hTNFα activity both in vitro and in vivo (see U.S. Pat.No. 6,090,382). Moreover, at least some of the antibodies of theinvention, such as D2E7, can neutralize hTNFα activity from otherspecies. Accordingly, the antibodies and antibody portions of theinvention can be used to inhibit hTNFα activity, e.g., in a cell culturecontaining hTNFα, in human subjects or in other mammalian subjectshaving TNFαs with which an antibody of the invention cross-reacts (e.g.chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). Inone embodiment, the invention provides a method for inhibiting TNFαactivity comprising contacting TNFα with an antibody or antibody portionof the invention such that TNFα activity is inhibited. Preferably, theTNFα is human TNFα. For example, in a cell culture containing, orsuspected of containing TNFα, an antibody or antibody portion of theinvention can be added to the culture medium to inhibit hTNFα activityin the culture.

[0104] In a preferred embodiment, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial, comprising subcutaneously administering to the subjectbiweekly an antibody or antibody portion of the invention such that thedisorder is treated. In a particularly preferred embodiment, theantibody is administered subcutaneously on a biweekly schedule. Inanother particularly preferred embodiment, the antibody is administeredsubcutaneously before, during or after administration of methotrexate.Preferably, the subject is a human subject. Alternatively, the subjectcan be a mammal expressing a TNFα with which an antibody of theinvention crossreacts. Still further the subject can be a mammal intowhich has been introduced hTNFα (e.g., by administration of hTNFα or byexpression of an hTNFα transgene). An antibody of the invention can beadministered to a human subject for therapeutic purposes (discussedfurther below). Moreover, an antibody of the invention can beadministered to a non-human mammal expressing a TNFα with which theantibody cross-reacts (e.g., a primate, pig or mouse) for veterinarypurposes or as an animal model of human disease. Regarding the latter,such animal models may be useful for evaluating the therapeutic efficacyof antibodies of the invention (e.g., testing of dosages and timecourses of administration).

[0105] As used herein, the term “a disorder in which the administrationof an anti-TNFα antibody is beneficial” is intended to include diseasesand other disorders in which the presence of TNFα in a subject sufferingfrom the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder, or where it has been shownthat another anti-TNFα antibody or a biologically active portion thereofhas been successfully used to treat the disease. Accordingly, a disorderin which α activity is detrimental is a disorder in which inhibition ofTNFα activity is expected to alleviate the symptoms and/or progressionof the disorder. Such disorders may be evidenced, for example, by anincrease in the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, forexample, using an anti-TNFα antibody as described above.There are numerous examples of disorders in which TNFα activity isdetrimental. The use of the antibodies and antibody portions of theinvention in the treatment of specific disorders is discussed furtherbelow:

[0106] A. Sepsis

[0107] Tumor necrosis factor has an established role in thepathophysiology of sepsis, with biological effects that includehypotension, myocardial suppression, vascular leakage syndrome, organnecrosis, stimulation of the release of toxic secondary mediators andactivation of the clotting cascade (see e.g., Tracey, K. J. and Cerami,A. (1994) Annu. Rev. Med. 45:491-503; Russell, D and Thompson, R. C.(1993) Curr. Opin. Biotech. 4:714-721). Accordingly, the humanantibodies, and antibody portions, of the invention can be used to treatsepsis in any of its clinical settings, including septic shock,endotoxic shock, gram negative sepsis and toxic shock syndrome.

[0108] Furthermore, to treat sepsis, an anti-hTNFα antibody, or antibodyportion, of the invention can be coadministered with one or moreadditional therapeutic agents that may further alleviate sepsis, such asan interleukin-1 inhibitor (such as those described in PCT PublicationNos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g.,PCT Publication No. WO 93/11793) or an antagonist of platelet activatingfactor (see e.g., European Patent Application Publication No. EP 374510).

[0109] Additionally, in a preferred embodiment, an anti-TNFα antibody orantibody portion of the invention is administered to a human subjectwithin a subgroup of sepsis patients having a serum or plasmaconcentration of IL-6 above 500 pg/ml, and more preferably 1000 pg/ml,at the time of treatment (see PCT Publication No. WO 95/20978 by Daum,L., et al.).

[0110] B. Autoimmune Diseases

[0111] Tumor necrosis factor has been implicated in playing a role inthe pathophysiology of a variety of autoimmune diseases. For example,TNFα has been implicated in activating tissue inflammation and causingjoint destruction in rheumatoid arthritis (see e.g., Tracey and Cerami,supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum. 38:151-160;Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFα alsohas been implicated in promoting the death of islet cells and inmediating insulin resistance in diabetes (see e.g., Tracey and Cerami,supra; PCT Publication No. WO 94/08609). TNFα also has been implicatedin mediating cytotoxicity to oligodendrocytes and induction ofinflammatory plaques in multiple sclerosis (see e.g., Tracey and Cerami,supra). Chimeric and humanized murine anti-hTNFα antibodies haveundergone clinical testing for treatment of rheumatoid arthritis (seee.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J.,et al. (1994) Lancet 344:1105-1110; Rankin, E. C., et al. (1995) Br. J.Rheumatol. 34:334-342).

[0112] The human antibodies, and antibody portions of the invention canbe used to treat autoimmune diseases, in particular those associatedwith inflammation, including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis and nephroticsyndrome. Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion at a site of inflammation may bebeneficial (e.g., local administration in the joints in rheumatoidarthritis or topical application to diabetic ulcers, alone or incombination with a cyclohexane-ylidene derivative as described in PCTPublication No. WO 93/19751).

[0113] C. Infectious Diseases

[0114] Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria (see e.g., Tracey and Cerami,supra). TNFα also has been implicated in mediating brain inflammation,inducing breakdown of the blood-brain barrier, triggering septic shocksyndrome and activating venous infarction in meningitis (see e.g.,Tracey and Cerami, supra). TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS) (see e.g.,Tracey and Cerami, supra). Accordingly, the antibodies, and antibodyportions, of the invention, can be used in the treatment of infectiousdiseases, including bacterial meningitis (see e.g., European PatentApplication Publication No. EP 585 705), cerebral malaria, AIDS andAIDS-related complex (ARC) (see e.g., European Patent ApplicationPublication No. EP 230 574), as well as cytomegalovirus infectionsecondary to transplantation (see e.g., Fietze, E., et al. (1994)Transplantation 58:675-680). The antibodies, and antibody portions, ofthe invention, also can be used to alleviate symptoms associated withinfectious diseases, including fever and myalgias due to infection (suchas influenza) and cachexia secondary to infection (e.g., secondary toAIDS or ARC).

[0115] D. Transplantation

[0116] Tumor necrosis factor has been implicated as a key mediator ofallograft rejection and graft versus host disease (GVHD) and inmediating an adverse reaction that has been observed when the ratantibody OKT3, directed against the T cell receptor CD3 complex, is usedto inhibit rejection of renal transplants (see e.g., Tracey and Cerami,supra; Eason, J. D., et al. (1995) Transplantation 59:300-305;Suthanthiran, M. and Strom, T. B. (1994) New Engl. J. Med. 331:365-375).Accordingly, the antibodies, and antibody portions, of the invention,can be used to inhibit transplant rejection, including rejections ofallografts and xenografts and to inhibit GVHD. Although the antibody orantibody portion may be used alone, more preferably it is used incombination with one or more other agents that inhibit the immuneresponse against the allograft or inhibit GVHD. For example, in oneembodiment, an antibody or antibody portion of the invention is used incombination with OKT3 to inhibit OKT3-induced reactions. In anotherembodiment, an antibody or antibody portion of the invention is used incombination with one or more antibodies directed at other targetsinvolved in regulating immune responses, such as the cell surfacemolecules CD25 (interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1),CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet anotherembodiment, an antibody or antibody portion of the invention is used incombination with one or more general immunosuppressive agents, such ascyclosporin A or FK506.

[0117] E. Malignancy

[0118] Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies (see e.g., Tracey and Cerami, supra).Accordingly, the antibodies, and antibody portions, of the invention,can be used in the treatment of malignancies, to inhibit tumor growth ormetastasis and/or to alleviate cachexia secondary to malignancy. Theantibody, or antibody portion, may be administered systemically orlocally to the tumor site.

[0119] F. Pulmonary Disorders

[0120] Tumor necrosis factor has been implicated in the pathophysiologyof adult respiratory distress syndrome, including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome (see e.g., Tracey and Cerami,supra). Accordingly, the antibodies, and antibody portions, of theinvention, can be used to treat various pulmonary disorders, includingadult respiratory distress syndrome (see e.g., PCT Publication No. WO91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonarysarcoidosis, pulmonary fibrosis and silicosis. The antibody, or antibodyportion, may be administered systemically or locally to the lungsurface, for example as an aerosol.

[0121] G. Intestinal Disorders

[0122] Tumor necrosis factor has been implicated in the pathophysiologyof inflammatory bowel disorders (see e.g., Tracy, K. J., et al. (1986)Science 234:470-474; Sun, X-M., et al. (1988) J. Clin. Invest.81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.81:301-305). Chimeric murine anti-hTNFα antibodies have undergoneclinical testing for treatment of Crohn's disease (van Dullemen, H. M.,et al. (1995) Gastroenterology 109:129-135). The human antibodies, andantibody portions, of the invention, also can be used to treatintestinal disorders, such as idiopathic inflammatory bowel disease,which includes two syndromes, Crohn's disease and ulcerative colitis.

[0123] H. Cardiac Disorders

[0124] The antibodies, and antibody portions, of the invention, also canbe used to treat various cardiac disorders, including ischemia of theheart (see e.g., European Patent Application Publication No. EP 453 898)and heart insufficiency (weakness of the heart muscle) (see e.g., PCTPublication No. WO 94/20139).

[0125] I. Others

[0126] The antibodies, and antibody portions, of the invention, also canbe used to treat various other disorders in which TNFα activity isdetrimental. Examples of other diseases and disorders in which TNFαactivity has been implicated in the pathophysiology, and thus which canbe treated using an antibody, or antibody portion, of the invention,include inflammatory bone disorders and bone resorption disease (seee.g., Bertolini, D. R., et al. (1986) Nature 319:516-518; Konig, A., etal. (1988) J. Bone Miner. Res. 3:621-627; Lerner, U. H. and Ohlin, A.(1993) J. Bone Miner. Res. 8:147-155; and Shankar, G. and Stern, P. H.(1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (seee.g., McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351;Felver, M. E., et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; andHansen, J., et al. (1994) Hepatology 20:461-474) and viral hepatitis(Sheron, N., et al. (1991) J. Hepatol. 12:241-245; and Hussain, M. J.,et al. (1994) J. Clin. Pathol. 47:1112-1115), coagulation disturbances(see e.g., van der Poll, T., et al. (1990) N. Engl. J. Med.322:1622-1627; and van der Poll, T., et al. (1991)Prog. Clin. Biol. Res.367:55-60), burns (see e.g., Giroir, B. P., et al. (1994) Am. J.Physiol. 267:H118-124; and Liu, X. S., et al. (1994) Burns 20:40-44),reperfusion injury (see e.g., Scales, W. E., et al. (1994) Am. J.Physiol. 267:G1122-1127; Serrick, C., etal. (1994 ) Transplantation58:1158-1162; and Yao, Y. M., et al. (1995) Resuscitation 29:157-168),keloid formation (see e.g., McCauley, R. L., et al. (1992) J. Clin.Immunol. 12:300-308), scar tissue formation and pyrexia.

[0127] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLE 1 Treatment with an Anti-TNFα Antibody D2E7 Efficacy FollowingSubcutaneous Administration

[0128] In this study, twenty-four patients with active RA were treatedwith weekly doses of 0.5 mg/kg D2E7 (n=18) or placebo (n=6) by s.c.injection for three months. Patients participating in this study had amean duration of disease of 10.1 years with a disease activity score(DAS) score of 4.87 and a mean of 3.4 DMARDs (disease modifyinganti-rheumatic drugs) prior to study entry; again reflectingconsiderable disease activity. Responders continued open-label treatmentwith D2E7, while patients who failed to respond to the 0.5 mg/kg dose orwho lost a DAS response on the 0.5 mg/kg dose were escalated to receive1 mg/kg by s.c. injection after week twelve of the study.

[0129] The first patients enrolled received up to sixty injections andwere, therefore, sixty weeks on the study drug. The efficacy with s.c.dosing was similar to i.v. injections. Up to 78% of patients reached aDAS and ACR20response during the first weeks of treatment. SubcutaneousD2E7 at a dose of 0.5 mg/kg/week reduced the swollen joint (SWJ) countby 54%, tender joint count (TJC) by 61% and CRP by 39% over twelve weekscompared to baseline, whereas all parameters increased in the placebogroup. After completion of the placebo-controlled period of this study,the patients continued treatment for up to fourteen months withsustained efficacy. These results indicate that subcutaneous D2E7 at adose of 0.5 mg/kg/week can, therefore, be safely self-administered withgood local tolerability.

[0130] Administration of D2E7 and Methotrexate

[0131] In this study, patients received s.c. or i.v. placebo or D2E7 ata dose of 1 mg/kg in addition to their ongoing treatment with(methotrexate) MTX. Fifty-four patients were enrolled in the study andeighteen patients received i.v. D2E7 and s.c. placebo, eighteen patientsreceived i.v. placebo and s.c. D2E7, and eighteen patients receivedplacebo i.v. and s.c. The patients received their second dose only afterthey lost their blinded response status, not earlier than four weeksafter the first dose. Thereafter, all patients received open-labelbiweekly s.c. injections of D2E7.

[0132] Demographic characteristics of the study population of this studyincluded a mean duration of RA of 11.1 years, prior exposure to a meanof 3.6 DMARDs (other than MTX), and a mean DAS at study entry of 4.81.By Day twenty-nine, 72% of the i.v. D2E7 treated patients and 44% of thes.c. D2E7 treated patients had achieved a response by DAS criteria,compared to only 28% of placebo-treated patients (set forth in FIG. 5).Of the responders in this study, 28% of placebo treated patientsmaintained an ACR20 response up to day 29, compared to 72% ofi.v.-treated D2E7 patients and 67% of s.c.-treated D2E7 patients, whomaintained their responses for between one and three months.

EXAMPLE 2 Total Body Dose of a Subcutaneously Administered Anti-TNFαAntibody

[0133] Weekly, Subcutaneous Administration of D2E7

[0134] This study enrolled two hundred eighty-four patients with RA andwas designed to determine the optimal total body dose of subcutaneouslyadministered D2E7. Patients were randomized to receive either 20, 40, or80 mg D2E7 or placebo weekly for twelve weeks, after which timeplacebo-treated patients were switched blindly to 40 mg D2E7/week.

[0135] Approximately 49% of patients reached ACR20 at 20 mg, 55% ofpatients reached ACR20 at 40 mg, and 54% of patients reached ACR20 at 80mg, while only 10% of patients receiving placebo reached ACR20 (setforth in FIG. 1A). Approximately 23% of patients reached ACR50 at 20 mg,27% of patients reached ACR50 at 40 mg, and 20% of patients reachedACR50 at 80 mg, and only 2% of patients receiving placebo reached ACR50.These data illustrate that subcutaneous D2E7, particularly at a dose of40 mg/week, generates a good response.

EXAMPLE 3 Biweekly, Subcutaneous Administration of an Anti-TNFα AntibodyBiweekly, Subcutaneous Administration of D2E7

[0136] The clinical effects, safety, immunogenicity, and tolerance of RApatients with partial responses to MTX following every other weeksubcutaneous (s.c.) injections of placebo or D2E7 at several dose levelsfor up to twenty-four weeks in conjunction with continued MTX treatmentwas investigated.

[0137] Study Design

[0138] A placebo-controlled, double-blind, randomized, multi-centerstudy in patients with RA, who had insufficient efficacy or tolerabilityto MTX was performed. During the course of the trial, patients werecontinued on a stable dose of MTX with dose ranges specified in theinclusion criteria described below.

[0139] This study consisted of two portions: 1) a “wash-out period” offour weeks prior to the administration of the first dose medication,during which time DMARDs (except for MTX) were withdrawn; and 2) a“placebo controlled period” during which time patients were randomizedto one of four cohorts of sixty-seven patients to receive placebo, 20,40, or 80 mg D2E7 (as a total body dose) given every other week s.c. forup to 24 weeks. Each dose of study drug was administered as two s.c.injections of 1.6 mL each. The patient's first dose was administered bymedical personnel as part of the patient's training. Subsequent doseswere self-administered by the patient at the study under the directobservation of trained personnel for the first four weeks. Thereafter,doses were administered outside the study site by the patient, a trainedindividual designated by the patient, or by medical personnel.Medication for four or five weeks was dispensed after each clinicalassessment. Patients were serially examined in weeks one, two, three,four, six, eight, twelve, sixteen, twenty, and twenty-four of the studywith the joint examinations being performed by a blinded assessor,independent of the treating physician.

[0140] This study enrolled two hundred seventy-one patients with RA. Thestudy population was representative of the moderate to severe RApopulation in North America: approximately 70% female, and predominantlyover the age of forty. The population was selected using predeterminedinclusion and exclusion criteria, known to those of skill in the arte.g., a patient must have received a diagnosis of RA as defined by the1987-revised American College of Rheumatology (ACR) criteria (set forthin Appendix A)

[0141] Results

[0142] FIGS. 1B and 2-4 indicate that subcutaneous, biweekly D2E7treatment combined with methotrexate was significantly better thanplacebo in reducing the signs and symptoms of RA at twenty-four weeks.All three doses of D2E7 were statistically significantly more effectivethan placebo given weekly. Furthermore, D2E7 at 40 mg and 80 mg hadbetter efficacy than the 20 mg dose.

[0143] Equivalents

[0144] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed:
 1. A method for treating a disorder in a human subjectin which the disease is treatable with a TNFα antibody, comprisingadministering a composition to the human subject in need thereof, on abiweekly dosing regimen such that the disorder is treated, saidcomposition containing an anti-TNFα antibody or an antigen bindingportion thereof.
 2. The method of claim 1, wherein the administration isby subcutaneous injection.
 3. The method of claim 1, wherein saidanti-TNFα antibody or an antigen binding portion thereof is a humananti-TNFα antibody.
 4. The method of claim 3, wherein said humanantibody, or an antigen-binding portion thereof, dissociates from humanTNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ Of 1×10⁻⁷ M orless.
 5. The method of claim 3, wherein saidhuman antibody, or antigen-binding portion thereof, dissociates fromhuman TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹ or less.
 6. Themethod of claim 3, wherein said human antibody, or antigen-bindingportion thereof, dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻⁴ s⁻¹ or less.
 7. The method of claim 3, wherein saidhuman antibody, or antigen-binding portion thereof, neutralizes humanTNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of1×10⁻⁸ M or less.
 8. The method of claim 3, wherein said human antibody,or antigen-binding portion thereof, neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ M or less. 9.The method of claim 3, wherein said human antibody, or antigen-bindingportion thereof, neutralizes human TNFα cytotoxicity in a standard invitro L929 assay with an IC₅₀ of 1×10⁻¹⁰ M or less.
 10. The method ofclaim 3, wherein said human antibody, or antigen-binding portionthereof, is a recombinant antibody, or recombinant antigen-bindingportion thereof.
 11. A method for inhibiting human TNFα activity in ahuman subject suffering from a disorder in which TNFα activity isdetrimental, comprising administering a composition to the human subjecton a biweekly dosing regimen, said composition containing a humananti-TNFα antibody wherein said human antibody, or antigen-bindingportion thereof, has the following characteristics: a) dissociates fromhuman TNFα with a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance; b) has a light chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8 or by one to five conservative amino acid substitutions atpositions 1, 3, 4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11 or by one to five conservative amino acid substitutions atpositions 2, 3, 4, 5, 6, 8,9, 10, 11 and/or
 12. 12. The method of claim11, wherein the administering is subcutaneously.
 13. The method of claim11, wherein said human antibody, or an antigen-binding portion thereof,dissociates from human TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹or less.
 14. A method for inhibiting human TNFα activity in a humansubject suffering from a disorder in which TNFα activity is detrimental,comprising administering a composition to the human subject,subcutaneously on a biweekly dosing regimen, said composition containinga human anti-TNFα antibody wherein said human antibody, or anantigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or
 11. 15. The method of claim 14,wherein the LCVR of said human antibody, or an antigen-binding portionthereof, further has a CDR2 domain comprising the amino acid sequence ofSEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the aminoacid sequence of SEQ ID NO:
 6. 16. The method of claim 14, wherein theLCVR of said human antibody, or an antigen-binding portion thereof,further has CDR1 domain comprising the amino acid sequence of SEQ ID NO:7 and the HCVR has a CDR1 domain comprising the amino acid sequence ofSEQ ID NO:
 8. 17. A method for inhibiting human TNFα activity in a humansubject suffering from a disorder in which TNFα activity is detrimental,comprising administering a composition to the human subject,subcutaneously on a biweekly dosing regimen, said composition containinga human anti-TNFα antibody wherein said human antibody, or an antigenbinding portion thereof, has a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2.
 18. The method of claim 17, wherein said human antibody has an IgGIheavy chain constant region.
 19. The method of claim 17, wherein saidhuman antibody has an IgG4 heavy chain constant region.
 20. The methodof claim 17, wherein said human antibody is a Fab fragment.
 21. Themethod of claim 17, wherein said human antibody is a single chain Fvfragment.
 22. A method for inhibiting human TNFα activity in a humansubject suffering from a disorder in which TNFα activity is detrimental,comprising administering a composition to the human subject,subcutaneously on a biweekly dosing regimen, said composition containinga human anti-TNFα antibody wherein said human antibody, or anantigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 orhas a heavy chain variable region (HCVR) having a CDR3 domain comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO:
 34. 23. A method forinhibiting human TNFα activity in a human subject suffering from adisorder in which TNFα activity is detrimental, comprising administeringa composition to the human subject, subcutaneously on a biweekly dosingregimen, said composition containing a human anti-TNFα antibody whereinsaid human antibody is the antibody D2E7 or an antigen-binding portionthereof.
 24. A kit containing a formulation comprising: a) apharmaceutical composition comprising an anti-TNFα antibody and apharmaceutically acceptable carrier; and b) instructions for biweeklydosing of the pharmaceutical composition for the treatment of a disorderin which an anti-TNFα antibody or a binding portion thereof is effectivein treating the disorder.
 25. The kit of claim 24, wherein said antibodycomprises the antibody, or antigen-binding portion thereof, selectedfrom the group consisting of: an antibody, or antigen-binding portionthereof that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less; anantibody, or antigen-binding portion thereof that dissociates from humanTNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(off) rate constant of 1×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less; anantibody, or antigen-binding portion thereof that neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ Mor less; an antibody, or antigen-binding portion thereof thatneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻¹⁰ M or less; an antibody, or antigen-bindingportion thereof that a) dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, as determined by surface plasmonresonance; b) has a light chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3 , or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8 or by one to fiveconservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9; c) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12; an antibody, or antigen-binding portionthereof that is a human anti-TnFα antibody wherein said human antibody,or an antigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein the LCVR of saidhuman antibody, or an antigen-binding portion thereof, having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, and has a heavy chain variable region (HCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modifiedfrom SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4,5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 6; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinthe LCVR of said human antibody, or an antigen-binding portion thereof,having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3,or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6; and has CDR1domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVRhas a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen binding portion thereof, has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2, and wherein said human antibody has anIgG1 heavy chain constant region; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein said humanantibody, or an antigen binding portion thereof, has a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2, and wherein said human antibody has an IgG4heavy chain constant region; an antibody, or antigen-binding portionthereof is a human anti-TNFα antibody wherein said human antibody, or anantigen binding portion thereof, has a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2, and wherein said human antibody is a Fab fragment; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2, and wherein saidhuman antibody is a single chain Fv fragment; and an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen-binding portion thereof, has a lightchain variable region (LCVR) having a CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26 or has a heavy chain variable region (HCVR) having aCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ IDNO:
 34. 26. A preloaded syringe containing a pharmaceutical compositioncomprising an anti-TNFα antibody and a pharmaceutically acceptablecarrier.
 27. The syringe of claim 26, wherein said human antibodycomprises the antibody, or antigen-binding portion thereof selected fromthe group consisting of: an antibody, or antigen-binding portion thereofthat dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less; an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(off) rate constant of 5×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(off) rate constant of 1×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less; anantibody, or antigen-binding portion thereof that neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ Mor less; an antibody, or antigen-binding portion thereof thatneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻¹⁰ M or less; an antibody, or antigen-bindingportion thereof that a) dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, as determined by surface plasmonresonance; b) has a light chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8 or by one to fiveconservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9; c) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12; an antibody, or antigen-binding portionthereof that is a human anti-TNFα antibody wherein said human antibody,or an antigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein the LCVR of saidhuman antibody, or an antigen-binding portion thereof, having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, and has a heavy chain variable region (HCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modifiedfrom SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4,5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 6; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinthe LCVR of said human antibody, or an antigen-binding portion thereof,having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3,or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6; and has CDR1domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVRhas a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen binding portion thereof, has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2, and wherein said human antibody has anIgG1 heavy chain constant region; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein said humanantibody, or an antigen binding portion thereof, has a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2, and wherein said human antibody has an IgG4heavy chain constant region; an antibody, or antigen-binding portionthereof is a human anti-TNFα antibody wherein said human antibody, or anantigen binding portion thereof, has a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2, and wherein said human antibody is a Fab fragment; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2, and wherein saidhuman antibody is a single chain Fv fragment; and an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen-binding portion thereof, has a lightchain variable region (LCVR) having a CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26 or has a heavy chain variable region (HCVR) having aCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ IDNO:
 34. 28. A method for treating a disorder in which an anti-TNFαantibody or antigen binding portion thereof is effective in treating thedisorder, comprising administering to the human subject subcutaneously acomposition containing an anti-TNFα antibody or an antigen bindingportion thereof and methotrexate such that the disorder is treated. 29.The method of claim 28, wherein methotrexate is administered togetherwith the administration of an anti-TNFα antibody or antigen bindingportion thereof.
 30. The method of claim 28, wherein methotrexate isadministered prior to the administration of an anti-TNFα antibody orantigen binding portion thereof.
 31. The method of claim 28, whereinmethotrexate is administered after the administration of an anti-TNFαantibody or antigen binding portion thereof.
 32. The method of claim 28,wherein said anti-TNFα antibody is a human anti-TNFα antibody or antigenbinding portion thereof.
 33. The method of claim 28, wherein said humanantibody, or an antigen-binding portion thereof, dissociates from humanα with a K_(d) Of 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less.
 34. The method of claim 28, whereinsaid human antibody, or antigen-binding portion thereof, dissociatesfrom human TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹ or less. 35.The method of claim 28, wherein said human antibody, or antigen-bindingportion thereof, dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻⁴ s⁻¹ or less.
 36. The method of claim 28, wherein saidhuman antibody, or antigen-binding portion thereof, neutralizes humanTNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of1×10⁻⁸ M or less.
 37. The method of claim 28, wherein said humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ Mor less.
 38. The method of claim 28, wherein said human antibody, orantigen-binding portion thereof, neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻¹⁰ M or less. 39.The method of claim 28, wherein said human antibody, or antigen-bindingportion thereof, is a recombinant antibody, or antigen-binding portionthereof.
 40. The method of claim 28, wherein said human antibody, orantigen-binding portion thereof, inhibits human TNFα-induced expressionof ELAM-1 on human umbilical vein endothelial cells.
 41. A method forinhibiting human TNFα activity in a human subject suffering from adisorder in which TNFα activity is detrimental, comprising administeringto the human subject methotrexate and subcutaneously, a human anti-TNFαantibody wherein said human antibody, or antigen-binding portionthereof, has the following characteristics: a) dissociates from humanTNFα with a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, as determinedby surface plasmon resonance; b) has a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 orby one to five conservative amino acid substitutions at positions 1, 3,4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domain comprising theamino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by asingle alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 orby one to five conservative amino acid substitutions at positions 2, 3,4, 5, 6, 8, 9, 10, 11 and/or
 12. 42. The method of claim 41, whereinsaid human antibody, or an antigen-binding portion thereof, dissociatesfrom human TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹ or less. 43.The method of claim 41, wherein said human antibody, or anantigen-binding portion thereof, dissociates from human TNFα with aK_(off) rate constant of 1×10⁻⁴ s⁻¹ or less.
 44. A method for inhibitinghuman TNFα activity in a human subject suffering from a disorder inwhich TNFα activity is detrimental, comprising administering to thehuman subject methotrexate and subcutaneously a human anti-TNFαantibody, wherein said human antibody, or an antigen-binding portionthereof, has a light chain variable region (LCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8,and has a heavy chain variable region (HCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or
 11. 45. The method of claim 44, wherein the LCVR of said humanantibody, or an antigen-binding portion thereof, further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 and the HCVRfurther has a CDR2 domain comprising the amino acid sequence of SEQ IDNO:
 6. 46. The method of claim 44, wherein the LCVR of said humanantibody, or an antigen-binding portion thereof, further has CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has aCDR1 domain comprising the amino acid sequence of SEQ ID NO:
 8. 47. Amethod for inhibiting human TNFα activity in a human subject sufferingfrom a disorder in which TNFα activity is detrimental, comprisingadministering to the human subject methotrexate and subcutaneously ahuman anti-TNFα antibody wherein said human antibody, or an antigenbinding portion thereof, has a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2.
 48. The method of claim 47, wherein said human antibody has an IgG1heavy chain constant region.
 49. The method of claim 47, wherein saidhuman antibody has an IgG4 heavy chain constant region.
 50. The methodof claim 47, wherein said human antibody is a Fab fragment.
 51. Themethod of claim 47, wherein said human antibody is a single chain Fvfragment.
 52. A method for inhibiting human TNFα activity in a humansubject suffering from a disorder in which TNFα activity is detrimental,comprising administering to the human subject methotrexate andsubcutaneously a human anti-TNFα antibody, or an antigen-binding portionthereof, which has a light chain variable region (LCVR) having a CDR3domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or has a heavy chainvariable region (HCVR) having a CDR3 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33and SEQ ID NO:
 34. 53. A method for inhibitinghuman TNFα activity in a human subject suffering from a disorder inwhich TNFα activity is detrimental, comprising administering to thehuman subject methotrexate and subcutaneously a human anti-TNFα antibodywherein said human antibody is the antibody D2E7 or an antigen-bindingportion thereof.
 54. A kit comprising a formulation comprising: a) apharmaceutical composition comprising an anti-TNFα antibody or anantigen binding portion thereof, methotrexate and a pharmaceuticallyacceptable carrier; and b) instructions for subcutaneous dosing to asubject of the pharmaceutical composition for the treatment of adisorder in which TNFα activity is detrimental.
 55. The kit of claim 54,wherein said antibody comprises the antibody, or antigenbinding portionthereof is selected from the group consisting of: an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ orless, both determined by surface plasmon resonance, and neutralizeshuman TNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀of 1×10⁻⁷ M or less; an antibody, or antigen-binding portion thereofthat dissociates from human TNFα with a K_(off) rate constant of 5×10⁻⁴s⁻¹ or less; an antibody, or antigen-binding portion thereof thatdissociates from human TNFα with a K_(off) rate constant of 1×10⁻⁴ s⁻¹or less; an antibody, or antigen-binding portion thereof thatneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁸ M or less; an antibody, or antigen-bindingportion thereof that neutralizes human TNFα cytotoxicity in astandard invitro L929 assay with an IC₅₀ of 1×10⁻⁹ M or less; an antibody, orantigen-binding portion thereof that neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10⁻¹⁰ M or less; anantibody, or antigen-binding portion thereof that a) dissociates fromhuman TNFα with a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance; b) has a light chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8 or by one to five conservative amino acid substitutions atpositions 1, 3, 4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11 or by one to five conservative amino acid substitutions atpositions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12; an antibody, orantigen-binding portion thereof that is a human anti-TNFα antibodywherein said human antibody, or an antigen-binding portion thereof, hasa light chain variable region (LCVR) having a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by asingle alanine substitution at position 1, 4, 5, 7 or 8, and has a heavychain variable region (HCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein the LCVR of said human antibody, or an antigen-bindingportion thereof, having a CDR3 domain comprising the amino acid sequenceof SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8, and has a heavy chain variableregion (HCVR) having a CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 and has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 and the HCVRfurther has a CDR2 domain comprising the amino acid sequence of SEQ IDNO: 6; an antibody, or antigen-binding portion thereof is a humananti-TNFα antibody wherein the LCVR of said human antibody, or anantigen-binding portion thereof, having a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by asingle alanine substitution at position 1, 4, 5, 7 or 8, and has a heavychain variable region (HCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 and has aCDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 and theHCVR further has a CDR2 domain comprising the amino acid sequence of SEQID NO: 6; and has CDR1 domain comprising the amino acid sequence of SEQID NO: 7 and the HCVR has a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 8; an antibody, or antigen-binding portionthereof is a human anti-TNFα antibody wherein said human antibody, or anantigen binding portion thereof, has a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2; an antibody, or antigen-binding portion thereof is a humananti-TNFα antibody wherein said human antibody, or an antigen bindingportion thereof, has a light chain variable region (LCVR) comprising theamino acid sequence of SEQ ID NO: 1 and a heavy chain variable region(HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and whereinsaid human antibody has an IgG1 heavy chain constant region; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2, and wherein saidhuman antibody has an IgG4 heavy chain constant region; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen binding portion thereof, has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2, and wherein said human antibody is a Fabfragment; an antibody, or antigen-binding portion thereof is a humananti-TNFα antibody wherein said human antibody, or an antigen bindingportion thereof, has a light chain variable region (LCVR) comprising theamino acid sequence of SEQ ID NO: 1 and a heavy chain variable region(HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and whereinsaid human antibody is a single chain Fv fragment; and an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen-binding portion thereof, has a lightchain variable region (LCVR) having a CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26 or has a heavy chain variable region (HCVR) having aCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ IDNO:
 34. 56. A preloaded syringe containing a pharmaceutical compositioncomprising an anti-TNFα antibody, methotrexate, and a pharmaceuticallyacceptable carrier.
 57. The syringe of claim 56, wherein said antibodycomprises the antibody or antigen-binding portion thereof selected fromthe group consisting of: an antibody, or antigen-binding portion thereofthat dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less; an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(off) rate constant of 5×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that dissociates from human TNFα with aK_(off) rate constant of 1×10⁻⁴ s⁻¹ or less; an antibody, orantigen-binding portion thereof that neutralizes human TNFα cytotoxicityin a standard in vitro L929 assay with an IC₅₀ of 1×10hu −8 M or less;an antibody, or antigen-binding portion thereof that neutralizes humanTNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of1×10⁻⁹ M or less; an antibody, or antigen-binding portion thereof thatneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻¹⁰ M or less; an antibody, or antigen-bindingportion thereof that a) dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, as determined by surface plasmonresonance; b) has a light chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8 or by one to fiveconservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9; c) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12; an antibody, or antigen-binding portionthereof that is a human anti-TNFα antibody wherein said human antibody,or an antigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3,4, 5, 6, 8, 9, 10 or 11; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein the LCVR of saidhuman antibody, or an antigen-binding portion thereof, having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, and has a heavy chain variable region (HCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modifiedfrom SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4,5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 6; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinthe LCVR of said human antibody, or an antigen-binding portion thereof,having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3,or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11 and has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6; and has CDR1domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVRhas a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2; an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen binding portion thereof, has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2, and wherein said human antibody has anIgG1 heavy chain constant region; an antibody, or antigen-bindingportion thereof is a human anti-TNFα antibody wherein said humanantibody, or an antigen binding portion thereof, has a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2, and wherein said human antibody has an IgG4heavy chain constant region; an antibody, or antigen-binding portionthereof is a human anti-TNFα antibody wherein said human antibody, or anantigen binding portion thereof, has a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2, and wherein said human antibody is a Fab fragment; anantibody, or antigen-binding portion thereof is a human anti-TNFαantibody wherein said human antibody, or an antigen binding portionthereof, has a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2, and wherein saidhuman antibody is a single chain Fv fragment; and an antibody, orantigen-binding portion thereof is a human anti-TNFα antibody whereinsaid human antibody, or an antigen-binding portion thereof, has a lightchain variable region (LCVR) having a CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26 or has a heavy chain variable region (HCVR) having aCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ IDNO:
 34. 58. The method of claims 1, wherein the disorder is sepsis. 59.The method of claims 1, wherein the antibody is administered to thehuman subject together with the cytokine interleukin-6 (IL-6) or isadministered to a human subject with a serum or plasma concentration ofIL-6 above 500 pg/ml.
 60. The method of claims 1, wherein the disorderis an autoimmune disease.
 61. The method of claim 60, wherein saidautoimmune disease is selected from the group consisting of rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis.62. The method of claim 61, wherein said autoimmune disease isrheumatoid arthritis.
 63. The method of claim 60, wherein saidautoimmune disease is selected from the group consisting of an allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis andnephrotic syndrome.
 64. The method of claims 1, wherein the disorder isan infectious disease.
 65. The method of claims 1, wherein the disorderis transplant rejection or graft-versus-host disease.
 66. The method ofclaims 1, wherein the disorder is a malignancy.
 67. The method of claims1, wherein the disorder is a pulmonary disorder.
 68. The method ofclaims 1, wherein the disorder is an intestinal disorder.
 69. The methodof claims 1, wherein the disorder is a cardiac disorder.
 70. The methodof claims 1, wherein the disorder is selected from the group consistingof inflammatory bone disorders, bone resorption disease, alcoholichepatitis, viral hepatitis, coagulation disturbances, bums, reperfusioninjury, keloid formation, scar tissue formation and pyrexia.
 71. A kitcomprising: a) a pharmaceutical composition comprising an anti-TNFαantibody or an antigen binding portion thereof and a pharmaceuticallyacceptable carrier; b) a pharmaceutical composition comprisingmethotrexate and a pharmaceutically acceptable carrier; and c)instructions for subcutaneous dosing to a subject of the anti-TNFαantibody pharmaceutical composition and dosing of the methotrexatepharmaceutical composition before, simultaneously or after the dosing ofthe anti-TNFα antibody pharmaceutical composition.
 72. A kit accordingto claim 71, wherein the anti-TNFα antibody or an antigen bindingportion thereof is D2E7 or an antigen binding portion thereof.